Calpain substrate specificity Understanding the substrate specificity of conventional calpains Hiroyuki Sorimachi1,*, Hiroshi Mamitsuka2, and Yasuko Ono1 1Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan 2Bioinformatics Center, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan *Correspondence to Hiroyuki Sorimachi, Ph.D.: Calpain Project, Department of Advanced Science for Biomolecules, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo156-8506, Japan Tel: +81-3-5316-3277; Fax: +81-3-5316-3163; E-mail: [email protected] Abstract a large superfamily of intracellular Ca2+-dependent Calpains are intracellular Ca2+-dependent Cys Cys proteases (Goll et al., 2003; Liu et al., 2008; proteases that play important roles in a wide range Sorimachi et al., 2011a; b; Ono & Sorimachi, of biological phenomena via the limited 2012) that play pivotal roles in a wide range of proteolysis of their substrates. Genetic defects in biological phenomena by mediating limited calpain genes cause lethality and/or functional proteolysis of their substrates. Thus, calpains deficits in many organisms, including humans. function as proteolytic processing enzymes. This is Despite their biological importance, the in contrast to the major intracellular degradative mechanisms underlying the action of calpains, proteolytic systems, consisting of eraser proteases particularly of their substrate specificities, remain such as proteasomes and lysosomal peptidases. largely unknown. Studies show that certain The specificity of the sequence preferences influence calpain substrate ubiquitin/proteasome-mediated proteolysis is recognition, and some properties of amino acids defined by the specific recognition and tagging of have been successfully related to substrate substrates by ubiquitin ligases, whereas the specificity and to the calpains’ 3D structure. The lysosomal peptidases generally function through full spectrum of this substrate specificity, however, autophagy, a largely non-specific degradation has not been clarified using standard sequence machinery (although specific autophagic analysis algorithms, e.g., the position-specific degradations occur within certain contexts). scoring-matrix method. More advanced Another major intracellular protease, caspase, bioinformatics techniques were used recently to shows strict specificity for Asp in P1 amino acid identify the substrate specificities of calpains and residues (aars). In contrast to all the above to develop a predictor for calpain cleavage sites, intracellular proteolytic systems, calpains show a demonstrating the potential of combining empirical more complex/ambiguous substrate specificity. data acquisition and machine learning. This review Calpains are specific, because the same substrates discusses the calpains’ substrate specificities, are always proteolyzed at the same positions under introducing the benefits of bioinformatics varying conditions; however, the rules governing applications. In conclusion, machine learning has this specificity are not understood. led to the development of useful predictors for Calpains have been identified in most calpain cleavage sites, although the accuracy of the eukaryotes (an intriguing exception is predictions still needs improvement. Machine Schizosaccharomyces pombe) and a few learning has also elucidated information about the eubacteria; these homologues have a variety of properties of calpains’ substrate specificities, domain structures and physiological roles. The including a preference for sequences over most studied calpains are ubiquitous mammalian secondary structures and the existence of a types known as μ-calpain and m-calpain, i.e., substrate specificity difference between two ‘conventional’ calpains. Each is composed of two similar conventional calpains, which has never distinct subunits: a large (~80 kDa) catalytic been indicated biochemically. subunit, CAPN1 (previously called μCL or calpain-1) in μ-calpain or CAPN2 (mCL or Keywords: calpain; multiple kernel learning; calpain-2) in m-calpain, and a smaller (~30 kDa) PSSM; support vector machine; structure-function regulatory subunit, CAPNS1 (30K or CAPN4), relationship; substrate specificity which is common to both conventional calpains. This review refers to calpain enzymes according to Introduction their subunit composition. Thus, μ-calpain and Calpains (Clan CA, family C02; EC 3.4.22.17) are m-calpain are referred to, respectively, as 1 Calpain substrate specificity CAPN1/S1 (short for CAPN1/CAPNS1) and species. CAPN2/S1. CAPN1 and CAPN2 have an identical Calpain substrates for in vitro activity assays domain structure: an N-terminal anchor-helix; and inhibitors protease core-domains 1 and 2 (PC1 and PC2, Calpains cause limited proteolysis of their respectively); a C2-domain-like (C2L) domain; substrates, mainly within inter-domain and a penta-EF-hand (PEF(L)) domain (Figure 1). unstructured regions. Two exceptions are casein The protease domain structure composed of PC1 and myelin basic protein, which are proteolyzed and PC2 is defined as ‘CysPc’ (No. cd00044 in the exhaustively by calpains, and casein is the most Conserved Domain Database of the National common substrate used in in-vitro calpain assays. Center for Biotechnology Information). CAPNS1 Some synthetic oligopeptides, in conjunction with is composed of a Gly-rich (GR) domain and a fluorescent probes, are also used as in-vitro penta-EF-hand (PEF(S)) domain, which is similar substrates (see Table 1). A major problem of using to a PEF(L) domain. The Ca2+-binding functional these substrates is that they are not calpain-specific. domains, PC1, PC2, C2L, and PEF(L)/(S), For example, SLY-MCA is a good substrate for respectively bind one, one, several, and four Ca cathepsin-L-like protease (Brady et al., 2000), ions. SLLVY-MCA is also cleaved by chymotrypsin and proteasomes (Ishiura et al., 1985), and Mammalian calpains BocLM-CMCA is cleaved by fiber cell globulizing Using the CysPc as the defining domain for aminopeptidase (Chandra et al., 2002). As short calpain homologues, 15 genes are identified in oligopeptides are generally poor substrates for human genome (Sorimachi et al., 2011b). Other calpains, some longer peptide substrates were vertebrates have one or more orthologs of each developed using calpain substrate sequences to human calpain species, which can be classified improve specificity and efficacy (Table 1). These according to their domain structure. CAPN3 substrates, however, are also proteolyzed by other (previously called p94 or calpain-3), CAPN8 proteases. (nCL-2), CAPN9 (nCL-4), CAPN11 (μ/mCL), and Calpastatin is a highly specific endogenous CAPN12–14 are similar to CAPN1 and 2 (Figure proteinaceous inhibitor of CAPN1/S1 and 1), and are collectively called ‘classical’ calpains. CAPN2/S1 (both are equally susceptible). The remaining large subunits (CAPN5 (hTRA-3), Calpastatin contains four inhibitory unit repeats CAPN6, CAPN7 (PalBH), CAPN10, CAPN15 that have varying inhibition efficacies (Figure 1). (SOLH), and CAPN16 (C6orf103)) are called Peptides (20–40mers) corresponding to ‘non-classical’ calpains, and are further divided calpastatin’s reactive sites are also used as into several subfamilies. CAPN5–7 and 10 are calpain-specific inhibitors (Table 2). Several categorized as the PalB subfamily, and contain low-molecular-weight inhibitors of conventional CysPc, C2L, and C2L/C2 domains (CAPN7 calpains, such as leupeptin and E-64, have been additionally contains a microtubule-interacting and reported, although they are much less transport (MIT) motif at the N-terminus). CAPN15 calpain-specific than calpastatin. They also inhibit belongs to the SOL subfamily, which contains other Cys proteases, including Cys cathepsins and Zn-finger motifs, CysPc, and a SOL-homology papain, as well as proteasomes and matrix (SOH) domain; CAPN16 contains only part of the metalloproteinase-2 (Ali et al., 2012) (Table 2). CysPc domain, i.e., PC1 but not PC2. PD150606, PD151746, and PD145305 bind PEF Expression patterns also provide good domains to inhibit calpains, although they are not classification criteria for mammalian calpains. specific for calpains (Van den Bosch et al., 2002) CAPN1, 2, 5, 7, 10, 13–16 are expressed in most and are less effective than calpeptin (Gerencser et tissues, whereas CAPN3 (skeletal muscle), CAPN6 al., 2009). Thus, it is necessary to use several (embryonic muscle and placenta), CAPN8/9 different inhibitors to determine whether calpains (gastrointestinal tract), CAPN11 (testis), and are involved in specific phenomena. CAPN12 (hair follicles) are more tissue/organ-specific. Defects in some ubiquitous Ca2+ and Calpain activation calpains cause early-stage lethality (Dutt et al., Mechanistic studies on calpain activation 2006; Takano et al., 2011), suggesting the dramatically progressed once their primary (Ohno importance of ubiquitous calpains in early et al., 1984) and 3D (Hosfield et al., 1999; Strobl development. By contrast, defects in tissue-specific et al., 2000) structures were determined. The latter calpains result in restricted dysfunctions like showed that, in inactive calpain, the conformations muscular dystrophy (Richard et al., 1995), of the PC1 and PC2 domains separate them from indicating specialized functions of these calpain 2 Calpain substrate specificity one another, thus maintaining the active site (duVerle et al., 2010; 2011) sites)